Ethylene, a gaseous plant hormone, is involved in the regulation of a number of plant processes ranging from growth and development to fruit ripening. As in animal systems, response of plants to disease not only involves static processes, but also involves inducible defense mechanisms. One of the earliest detectable event to occur during plant-pathogen interaction is a rapid increase in ethylene biosynthesis. Ethylene biosynthesis, in response to pathogen invasion, correlates with increased defense mechanisms, chlorosis, senescence and abscission. The molecular mechanisms underlying operation of ethylene action, however, are unknown. Nonetheless, ethylene produced in response to biological stress is known to regulate the rate of transcription of specific plant genes. A variety of biological stresses can induce ethylene production in plants including wounding, bacterial, viral or fungal infection as can treatment with elicitors, such as glycopeptide elicitor preparations (prepared by chemical extraction from fungal pathogen cells). Researchers have found, for example, that treatment of plants with ethylene generally increases the level of many pathogen-inducible "defense proteins", including .beta.-1,3-glucanase, chitinase, L-phenylalanine ammonia lyase, and hydroxyproline-rich glycoproteins. The genes for these proteins can be transcriptionally activated by ethylene and their expression can be blocked by inhibitors of ethylene biosynthesis. Researchers have also characterized a normal plant response to the production or administration of ethylene, as a so-called "triple response". The triple response involves inhibition of root and stem elongation, radial swelling of the stem and absence of normal geotropic response (diageotropism).
Ethylene is one of five well-established plant hormones. It mediates a diverse array of plant responses including fruit ripening, leaf abscission and flower senescence.
The pathway for ethylene biosynthesis has been established (FIG. 6). Methionine is converted to ethylene with S-adenylmethionine (SAM) and 1-aminocyclopropane-1-carboxylic acid (ACC) as intermediates. The production of ACC from SAM is catalyzed by the enzyme ACC synthase. Physiological analysis has suggested that this is the key regulatory step in the pathway, see Kende, Plant Physiol. 1989, 91, 1-4. This enzyme has been cloned from several sources, see Sato et al., PNAS, (USA) 1989, 86, 6621; Van Der Straeten et al., PNAS, (USA) 1990, 87, 4859-4863; Nakajima et al., Plant Cell Physiol. 1990, 29, 989. The conversion of ACC to ethylene is catalyzed by ethylene forming enzyme (EFE), which has been recently cloned (Spanu et al., EMBO J 1991, 10, 2007. Aminoethoxy-vinylglycine (AVG) and .alpha.-aminoisobutyric acid (AIB) have been shown to inhibit ACC synthase and EFE respectively. Ethylene binding is inhibited non-competitively by silver, and competitively by several compounds, the most effective of which is trans-cyclooctane. ACC synthase is encoded by a highly divergent gene family in tomato and Arabidopsis (Theologis, A., Cell 70:181 (1992)). ACC oxidase, which converts ACC to ethylene, is expressed constitutively in most tissues (Yang et al., Ann. Rev. Plant Physiol. 1984, 35, 155), but is induced during fruit ripening (Gray et al. Cell 1993 72, 427). It has been shown to be a dioxygenase belonging to the Fe2+/ascorbate oxidase superfamily (McGarvey et al., Plant Physiol. 1992, 98, 554).
Etiolated dicotyledonous seedlings are normally highly elongated and display an apical arch-shaped structure at the terminal part of the shoot axis; the apical hook. The effect of ethylene on dark grown seedlings, the triple response, was first described in peas by Neljubow in 1901, Neljubow, D., Pflanzen Beih. Bot. Zentralb., 1901, 10, 128. In Arabidopsis, a typical triple response consists of a shortening and radial swelling of the hypocotyl, an inhibition of root elongation and an exaggeration of the curvature of the apical hook (FIGS. 7 and 16). Etiolated morphology is dramatically altered by stress conditions which induce ethylene production the ethylene-induced "triple response" may provide the seedling with additional strength required for penetration of compact soils, see Harpham et al., Annals of Bot., 1991, 68, 55. Ethylene may also be important for other stress responses. ACC synthase gene expression and ethylene production is induced by many types of biological and physical stress, such as wounding and pathogen infection, see Boller, T., in The Plant Hormone Ethylene, A. K. Mattoo and J. C. Suttle eds., 293-314, 1991, CRC Press, Inc. Boca Raton and Yu, Y. et al., Plant Phys., 1979, 63, 589, Abeles et al. 1992 Second Edition San Diego, Calif. Academic Press; and Gray et al. Plant Mol Biol. 1992 19, 69.
A number of researchers have identified the interaction between Arabidopsis thaliana and Pseudomonas syringae bacteria; Whalen et al., "Identification of Pseudomonas syringae Pathogens of Arabidopsis and a Bacterial Locus Determining Avirulence on Both Arabidopsis and Soybean", The Plant Cell 1991, 3, 49, Dong et al., "Induction of Arabidopsis Defense Genes by Virulent and Avirulent Pseudomonas syringae Strains and by a Cloned Avirulence Gene", The Plant Cell 1991, 3, 61, and Debener et al., "Identification and Molecular Mapping of a Single Arabidopsis thaliana Locus Determining Resistance to a Phytopathogenic Pseudomonas syringae isolate", The Plant Journal 1991, 1, 289. P. syringae pv. tomato (Pst) strains are pathogenic on Arabidopsis. A single bacterial gene, avrRpt2, was isolated that controls pathogen avirulence on specific Arabidopsis host genotype Col-0.
Bent, A. F., et al., "Disease Development in Ethylene-Insensitive Arabidopsis thaliana Infected with Virulent and Avirulent Pseudomonas and Xanthomonas Pathogens", Molecular Plant-Microbe Interactions 1992, 5, 372; Agrios, G. N., Plant Pathology 1988, 126, Academic Press, San Diego; and Mussel, H., "Tolerance to Disease", page 40, in Plant Disease: An Advanced Treatise, Volume 5, Hotsfall, J. G. and Cowling, E. B., eds., 1980, Academic Press, New York, establish the art recognized definitions of tolerance, susceptibility, and resistance. Tolerance is defined for purposes of the present invention as growth of a pathogen in a plant where the plant does not sustain damage. Resistance is defined as the inability of a pathogen to grow in a plant and no damage to the plant results. Susceptibility is indicated by pathogen growth with plant damage.
Regardless of the molecular mechanisms involved, the normal ethylene response of a plant to pathogen invasion has been thought to have a cause and effect relationship in the ability of a plant to fight off plant pathogens. Plants insensitive in any fashion to ethylene were believed to be incapable of eliciting a proper defense response to pathogen invasion, and thus unable to initiate proper defense mechanisms. As such, ethylene insensitive plants were thought to be less disease tolerant.
The induction of disease responses in plants requires recognition of pathogens or pathogen-induced symptoms. In a large number of plant-pathogen interactions, successful resistance is observed when the plant has a resistance gene with functional specificity for pathogens that carry a particular avirulence gene. If the plant and pathogen carry resistance and avirulence genes with matched specificity, disease spread is curtailed and a hypersensitive response involving localized cell death and physical isolation of the pathogen typically occurs. In the absence of matched resistance and avirulence genes, colonization and tissue damage proceed past the site of initial infection and disease is observed.
A better understanding of plant pathogen tolerance is needed. Also needed is the development of methods for improving the tolerance of plants to pathogens, as well as the development of easy and efficient methods for identifying pathogen tolerant plants.
Genetic and molecular characterization of several gene loci and protein products is set forth in the present invention. The results will reveal interactions among modulatory components of the ethylene action pathway and provide insight into how plant hormones function. Thus, the quantity, quality and longevity of food, such as fruits and vegetables, and other plant products such as flowers, will be improved thereby providing more products for market in both developed and underdeveloped countries.